Antisense oligonucleotides are a new class of pharmaceuticals. In general, antisense refers to the use of small, synthetic oligonucleotides, with the same constituents as that found in human DNA or RNA. The antisense oligonucleotides are designed as a complementary sequence of a part of a gene they are targeting in order to be able to adhere to this sequence and inhibit gene expression. Gene expression is inhibited through hybridization of an antisense oligonucleotide to a specific messenger RNA (mRNA) sense target according to the Watson-Crick base pairing in which adenosine and thymidine (uracile in mRNA) or guanosine and cytidine interact through hydrogen bonding. Two mechanisms can account for these effects, the first being hybridization with impaired translation of targeted mRNA, the second being the induction of RNase H or similar enzymes with degradation of mRNA. A major advantage of this strategy is the specificity of action with the potential for less side effects and toxicity, especially when applied to the site of action (topical treatment). This therapeutic strategy could potentially be applied to any disease where an over-expression of one or several genes is believed to cause the presence or persistence of the disease. As a result, there have been numerous studies of antisense oligonucleotides as therapeutic agents for cancer and viral diseases.
Antisense oligonucleotides can be used to inhibit interleukin (IL)-6 receptor expression and thus the effects of the acute inflammatory mediator interleukin-6 on cells. Few studies have been conducted to assess whether antisense oligonucleotides can be employed to inhibit other receptors on cells that are involved in inflammation, including, but not limited to inflammation associated with asthma and inflammation associated with atopic diseases and allergy or on cancerous cells.
Asthma is a disease that affects 5 to 10% of the population that has doubled in prevalence in the last 25 years. This increase has been noted especially in infants after a viral infection of the airways (bronchiolitis), in children and in occupational induced asthma. The recurrent breathing problems associated with asthma are often triggered by allergens but the exact cause of asthma is not yet known. However, it is believed that agents such as viruses are involved in the perpetuation of the abnormal inflammation that is found in the airways of patients with asthma and thus the persistence of the disease.
For this reason the current recommendations for first line therapy of asthma is a potent anti-inflammatory medication such as those containing corticosteroids and anti-leukotrienes. Although this therapy is effective in many patients, some patients are resistant to corticosteroids. This medication is also a potent immunosuppressive with long term side effects and has not been shown to be effective in the prevention of allergy or asthma. Anti-leukotrienes have some effect in allergy and asthma but are not as effective as corticosteroids.
Several inflammatory mediators play a role in the appearance and perpetuation of inflammation in the airways of patients with asthma. Some mediators attract the inflammatory cells into the airways either through chemotaxis of eosinophils (the chemokines: RANTES, eotaxins 1, 2, 3, MCP-3, 4 that act mostly in asthmatic inflammation through a receptor called CCR3) or through endothelial cell activation (IL-4, -13). Other mediators cause the priming and increased survival of inflammatory cells in the airways (IL-3, -4, -5, GM-CSF). These mediators thus consist of either specific chemokines for eosinophils or of cytokines of the T helper lymphocyte type 2 phenotype (Th2: IL-3, -4, -5, -6, -9, -10, -13 and GM-CSF), (John A E. and Lukacs N W., 2003 Sarcoidosis Vasc Diffuse Lung Dis., 20:180-189; Blease et al., 2003, Expert Opin Emerg Drugs. 8:71-81). An improvement, in asthma and general respiratory inflammation, has been shown when there is a decrease in these inflammatory mediators in the airways.
Allergy is a hypersensitivity to an allergen causing an undesirable immune response. Allergy is a disease that is extremely prevalent, for example atopic rhinitis and conjunctivitis affect around 30% of the population. Allergy is characterized by abnormal IgE production and inflammation to an allergen. In the presence of IgE and allergen, effector cells, such as the mast cells degranulate and release inflammatory mediators leading to the recruitment of the same inflammatory cells that are found in asthma. In allergic rhinitis (i.e. hayfever), allergic conjunctivitis, nasal polyposis, chronic sinusitis and eczema, such as atopic dermatitis, one finds the same excess in inflammatory mediators as those present in asthma. IL-4 and IL-13 are necessary for the production of IgE and the induction of the cells with a Th2 phenotype (Barnes P J., 2003, Cytokine Growth Factor Rev. 14:511-522; Schuh et al., 2003, Cytokine Growth Factor Rev. 2003, 14:503-510). Atopic diseases is a generic name for allergic diseases which are developed by exposure to allergens, especially in individuals with a genetic propensity for being easily sensitized to allergens. Individuals having these predisposing factors easily develop an abnormal immune response to alimentary antigens and inhalants. Some specific examples of allergic diseases are bronchial asthma, a topic dermatitis, urticaria, allergic rhinitis, allergic conjunctivitis and allergic enterogastritis.
A neoplasm is an abnormal tissue growth that is uncontrollable and progressive. A malignant neoplasm is often characterized as a cancer. Cancer is the second leading cause of death in humans and is a general term for more than 100 diseases characterized by abnormal proliferation of immortalized cells. One of the mechanisms that is involved in the persistence and increase in these cells is by the release of growth factors that act through receptors and lead to cellular proliferation. Amongst these growth factors, GM-CSF has been shown to be an important growth factor for several tumour cells. The chemokine receptor CCR3 was recently characterized in malignant B lymphocytes recovered from patients with chronic lymphocytic leukemia (CLL) and with hairy cell leukemia (HCL), (Trentin et al., 2004, Blood, 104, 502-508). Indeed, the transactivation of Epidermal Growth Factor Receptor (EGFR) through CCR3 chemokine receptor was found to be a critical pathway that elicits MAP kinase activation and cytokine production in bronchial epithelial cells (Adachi et al., 2004, Biochem. Biophys. Res. Commun. 320, 292-396). The inhibition of proliferation of cancerous cells by blocking the receptors for growth factors and/or for chemokines, may be important in the therapy of certain cancers.
Eosinophils are a type of white blood cell. They are granular leukocytes with a nucleus that usually has two lobes connected by a slender thread of chromatin, and cytoplasm containing course, round granules that are uniform in size and stainable by eosin. Hypereosinophilia is characterized by an increased number of eosinophils, often associated with allergies, asthmas and infections.
Some use of oligonucleotides directed against specific nucleic acid sequences coding for receptors, in order to inhibit inflammatory reactions is known. PCT Application No. WO 99/66037 by Renzi describes antisense oligonucleotides that are used for treating and/or preventing asthma, allergy, hypereosinophilia, general inflammation and cancer. Specifically, the oligonucleotides of Renzi are directed against nucleic acid sequences coding for a CCR3 receptor, a common sub-unit of IL-4 and IL-3 receptors, or a common sub-unit of IL-3, IL-5 and GM-CSF receptors. Among others, an antisense oligonucleotide identified as 107A (5′-GGGTCTGCAGCGGGATGGT-S′) (SEQ ID NO: 43), directed against the common, beta ((β) sub-unit of the IL-3, IL-5 and GM-CSF receptor, is disclosed therein.
For potential clinical uses, antisense oligonucleotides should exhibit stability against degradation by serum and cellular nucleases, show low non-specific binding to serum and cell proteins, exhibit enhanced recognition of the target mRNA sequence, demonstrate cell-membrane permeability and elicited cellular nucleases when complexed with complementary mRNA. It is well documented that oligonucleotides containing natural sugars (D-ribose and D-2-deoxyribose) and phosphodiester (PO) linkages are rapidly degraded by serum and intracellular nucleases, which limit their utility as effective therapeutic agents. Chemical strategic modifications have been described for oligonucleotides in order to improve their stability and efficacy as therapeutic agents. The main chemical changes included, modification of the sugar moiety, the base moiety, and/or modification or replacement of the internucleotide phosphodiester linkage. To date the most widely studied analogues are the phosphorothioate (PS) oligodeoxynucleotides, in which one of the non-bridging oxygen atoms in the phosphodiester backbone is replaced with a sulfur (Eckstein F., 1985, Ann. Rev. Biochem., 54: 367-402). Several antisense oligonucleotide generations have been developed and used for in vitro and for in vivo studies (Goodchild J., 2004, Curr. Opin. Mol. Ther., 2004, 6:120-128; Urban E. and R. Noe C R., 2003, Farmaco. 58:243-258).
Recently, Renzi et al. described the use of 2′,6′-diaminopurine (DAP) and analogs thereof in nucleic molecules for anti-inflammatory compositions (PCT Application No. WO 03/004511 A2). Also described in this reference is the preparation of nucleic molecules having an increased in vivo physiological efficiency and a reduced toxicity as compared to oligonucleotides without DAP. Renzi et al. further teaches that DAP substitution is particularly useful in preparing oligonucleotides directed to pulmonary/respiratory diseases such as cystic fibrosis, asthma, chronic bronchitis, chronic obstructive lung disease, eosinophilic bronchitis, allergies, allergic rhinitis, pulmonary fibrosis, adult respiratory distress syndrome, sinusitis, respiratory syncytial virus or other viral respiratory tract infection and cancer.
It would be desirable to have further antisense oligonueleotides directed against at least one specific common receptor for either Th2 cytokines or receptors for mediators that attract cells that respond to Th2 cytokines, in order to inhibit the inflammatory reaction that is present in asthma or allergy and to inhibit neoplastic cell proliferation.
It would also be highly desirable to have further antisense oligonucleotides directed against nucleic acid sequences coding for receptors so that by inhibiting these receptors these oligonucleotides could be employed in the therapy and/or prevention of asthma, allergy, hypereosinophilia, general inflammation and cancer.